Detection of group a streptococcus

ABSTRACT

The invention provides methods to detect Group A Streptococcus (GAS) in biological samples using real-time PCR. Primers and probes for the detection of GAS are provided by the invention. Articles of manufacture containing such primers and probes for detecting GAS are further provided by the invention.

TECHNICAL FIELD

[0001] This invention relates to bacterial diagnostics, and moreparticularly to detection of β-hemolytic Group A Streptococcus (GAS).

BACKGROUND

[0002]Streptococcus pyogenes is a group A streptococcal gram-positivebacterium that is the etiological agent of several diseases in humans,including pharyngitis and/or tonsillitis, skin infections (impetigo,erysipelas, and other forms of pyoderma), acute rheumatic fever (ARF),scarlet fever (SF), poststreptococcal glomerulonephritis (PSGN), and atoxic shock-like syndrome (TSLS). On a global basis, ARF is the mostcommon cause of pediatric heart disease. For example, it is estimatedthat in India, more than six million school-aged children suffer fromrheumatic heart disease. In the United States, “sore throat” is thethird most common reason for physician office visits and S. pyogenes isrecovered from about 30% of children with this complaint. There areabout 25-35 million cases of streptococcal pharyngitis per year in theUnited States, responsible for about 1-2 billion dollars per year inhealth care costs.

SUMMARY

[0003] The invention provides for methods of identifying group Astreptococcus (GAS) in a biological sample. Primers and probes fordetecting GAS are provided by the invention, as are kits containing suchprimers and probes. Methods of the invention can be used to rapidlyidentify GAS nucleic acids from specimens for diagnosis of GASinfection. Using specific primers and probes, the methods includeamplifying and monitoring the development of specific amplificationproducts using real-time PCR.

[0004] In one aspect, the invention features a method for detecting thepresence or absence of Group A Streptococcus (GAS) in a biologicalsample from an individual. The method to detect GAS includes performingat least one cycling step, which includes an amplifying step and ahybridizing ste. The amplifying step includes contacting the sample witha pair of ptsI primers to produce a ptsI amplification product if a GASptsI nucleic acid molecule is present in the sample, and the hybridizingstep includes contacting the sample with a pair of ptsI probes.Generally, the members of the pair of ptsI probes hybridize to theamplification product within no more than five nucleotides of eachother. A first ptsI probe of the pair of ptsI probes is typicallylabeled with a donor fluorescent moiety and a second ptsI probe of thepair of ptsI probes is typically labeled with a corresponding acceptorfluorescent moiety. The method further includes detecting the presenceor absence of fluorescence resonance energy transfer (FRET) between thedonor fluorescent moiety of the first ptsI probe and the acceptorfluorescent moiety of the second ptsI probe. The presence of FRET isusually indicative of the presence of GAS in the biological sample, andthe absence of FRET is usually indicative of the absence of GAS in thebiological sample. The method can still further include determining themelting temperature between one or both of the ptsI probe(s) and theptsI amplification product. The melting temperature can confirm thepresence or the absence of the GAS.

[0005] A pair of ptsI primers generally includes a first ptsI primer anda second ptsI primer. The first ptsI primer can include the sequence5′-AAA TGC AGT AGA AAG CTT AGG-3′ (SEQ ID NO: 1), and the second ptsIprimer can include the sequence 5′-TGC ATG TAT GGG TTA TCT TCC-3′ (SEQID NO: 2). The first ptsI probe can include the sequence 5′-TTG CTG ATCCAG AAA TGA T-3′ (SEQ ID NO: 3), and the second ptsI probe can includethe sequence 5′-AGC CAG GTT AAA GAA ACG ATT CGC-3′ (SEQ ID NO: 4).

[0006] The members of the pair of ptsI probes can hybridize within nomore than two nucleotides of each other, or can hybridize within no morethan one nucleotide of each other. A representative donor fluorescentmoiety is fluorescein, and representative acceptor fluorescent moiety isselected from the group consisting of LC-Red 640, LC-Red 705, Cy5, andCy5.5.

[0007] In one aspect, the detecting step includes exciting thebiological sample at a wavelength absorbed by the donor fluorescentmoiety and visualizing and/or measuring the wavelength emitted by theacceptor fluorescent moiety. In another aspect, the detecting comprisesquantitating the FRET. In yet another aspect, the detecting step isperformed after each cycling step, and further, can be performed inreal-time.

[0008] Generally, the presence of the FRET within 50 cycles, or within40 cycles, or within 30 cycles, indicates the presence of a GASinfection in the individual. Representative biological samples includethroat swabs, tissues and bodily fluids.

[0009] The above-described methods can further include preventingamplification of a contaminant nucleic acid. Preventing amplificationcan include performing the amplification step in the presence of uraciland treating the biological sample with uracil-DNA glycosylase prior toa first amplification step. In addition, the ycling step can beperformed on a control sample. A control sample can include the GAS ptsInucleic acid molecule. Alternatively, such a control sample can beamplified using a pair of control primers and hybridized using a pair ofcontrol probes. The control primers and the control probes are usuallyother than the ptsI primers and the ptsI probes, respectively. A controlamplification product is produced if control template is present in thesample, and the control probes hybridize to the control amplificationproduct.

[0010] In another aspect of the invention, there are provided articlesof manufacture, including a pair of ptsI primers; a pair of ptsI probes;and a donor fluorescent moiety and a corresponding fluorescent moiety. Apair of ptsI primers generally includes a first ptsI primer and a secondptsI primer. A first ptsI primer can include the sequence 5′-AAA TGC AGTAGA AAG CTT AGG-3′ (SEQ ID NO: 1), and the second ptsI primer caninclude the sequence 5′-TGC ATG TAT GGG TTA TCT TCC-3′ (SEQ ID NO: 2). Apair of ptsI probes can include a first ptsI probe and a second ptsIprobe. A first ptsI probe can include the sequence 5′-TTG CTG ATC CAGAAA TGA T-3′ (SEQ ID NO: 3), and the second ptsI probe can include thesequence 5′-AGC CAG GTT AAA GAA ACG ATT CGC-3′ (SEQ ID NO: 4). Theprobes in such articles of manufacture can be labeled with a donorfluorescent moiety and with a corresponding acceptor fluorescent moiety.The articles of manufacture also can include a package label or packageinsert having instructions thereon for using the pair of ptsI primersand the pair of ptsI probes to detect the presence or absence of GAS ina biological sample.

[0011] In yet another aspect, the invention provides a method fordetecting the presence or absence of GAS in a biological sample from anindividual. Such a method includes performing at least one cycling step,wherein a cycling step comprises an amplifying step and a hybridizingstep. An amplifying step includes contacting the sample with a pair ofptsI primers to produce a ptsI amplification product if a GAS ptsInucleic acid molecule is present in the sample. A hybridizing stepincludes contacting the sample with a ptsI probe, wherein the ptsI probeis labeled with a donor fluorescent moiety and a corresponding acceptorfluorescent moiety. The method further includes detecting the presenceor absence of fluorescence resonance energy transfer (FRET) between thedonor fluorescent moiety and the acceptor fluorescent moiety of the ptsIprobe. The presence or absence of FRET is indicative of the presence orabsence of GAS in the sample. Amplification can employ a polymeraseenzyme having 5′ to 3′ exonuclease activity, and the donor and acceptorfluorescent moieties can be within no more than 5 nucleotides of eachother on the probe. In such a method, the ptsI probe can include anucleic acid sequence that permits secondary structure formation thatresults in spatial proximity between the donor and the acceptorfluorescent moiety. In the above-described methods, the acceptorfluorescent moiety can be a quencher.

[0012] In another aspect, the invention provides a method for detectingthe presence or absence of GAS in a biological sample from anindividual. Such a method includes performing at least one cycling step,wherein a cycling step comprises an amplifying step and a dye-bindingstep. An amplifying step includes contacting the sample with a pair ofptsI primers to produce a ptsI amplification product if a GAS ptsInucleic acid molecule is present in the sample. A dye-binding stepcomprises contacting the ptsI amplification product with a nucleic acidbinding dye. The method further includes detecting the presence orabsence of binding of the nucleic acid binding dye to the amplificationproduct. The presence of binding is usually indicative of the presenceof GAS in the sample, and the absence of binding is usually indicativeof the absence of GAS in the sample. Representative nucleic acid bindingdyes include SYBRGreenI®, SYBRGold®, and ethidium bromide. Such a methodcan further include determining the melting temperature between the ptsIamplification product and the nucleic acid binding dye. The meltingtemperature can confirm the presence or absence of the GAS.

[0013] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

[0014] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe drawings and detailed description, and from the claims.

DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is an alignment of ptsI nucleic acid sequences from theOklahoma University M1 strain (Ferretti et al., 2001, Proc. Natl. Acad.Sci. USA, 98:4658-63) and from 11 Group A Streptococcus (GAS) isolates.The location of the primers and probes used herein for the real-time PCRassay are shown.

DETAILED DESCRIPTION

[0016] A real-time PCR assay that is more sensitive than existing assaysis described herein for detecting GAS in a biological sample. Primersand probes for detecting GAS infections and articles of manufacturecontaining such primers and probes are provided by the invention. Theincreased sensitivity of the real-time PCR assay for detecting GAScompared to other methods, as well as the improved features of real-timePCR including sample containment and real-time detection of theamplified product, make feasible the implementation of this technologyfor routine diagnosis of GAS infections in the clinical laboratory.

[0017] β-Hemolytic Group A Streptococcus (GAS)

[0018] Streptococci are Gram-positive, non-motile bacteria that areoften arranged in pairs or chains. Streptococci generally exist ascommensals and parasites in humans, animals, and saprophytes. Moststreptococcal strains are facultative anaerobes with complex nutritionalrequirements. Streptococcal strains typically require blood- orserum-enriched media for growth. Streptococcal strains areoxidase-negative and catalase-negative, the latter being useful fordistinguishing streptococci from staphylococci. Streptococci have arigid cell wall with a typical Gram-positive peptidoglycan layer, aninner plasma membrane, mesosomal vesicles, and a nucleoid. The cell wallis divided by crosswall septation.

[0019] The cell wall of GAS organisms contains group- and type-specificantigens. For example, GAS organisms produce a group-specificcarbohydrate (i.e., a C-polysaccharide) that is a branched polymer ofL-rhamnose and N-acetyl-D-glucosamine in a 2:1 ratio. TheN-acetyl-D-glucosamine is the antigenic component of the group-specificcarbohydrate. The carbohydrate is linked by phosphate-containing bridgesto peptidoglycans composed of N-acetyl-D-glucosamine, N-acetyl-D-muramicacid, D-glutamic acid, L-lysine, and D- and L-alanine. The GAS-specificcarbohydrate generally comprises 10% of the dry weight of the cell. Inaddition, GAS organisms produce two major classes of type-specificproteins, the M and the T antigens (minor classes include F, R, andM-like antigens). The M proteins are fimbriae-like extensions associatedwith virulent strains, while the T proteins are a useful epidemiologicalmarker that have not been associated with virulence. GAS organisms alsocontain a capsular polysaccharide composed of hyaluronic acid.

[0020] GAS Nucleic Acids and Oligonucleotides

[0021] A metabolic pathway chart showing thephosphoenolpyruvate:phosphotransferase system is available athttp://www.genome.ad.ip/kegg/pathway/eco/eco02060.html. Briefly, thephosphoenolpyruvate:phosphotransferase system (pep:pts or pts) iscomposed of two enzymes, HPr and enzyme I (or EI) encoded by the ptsHand ptsI genes, respectively. Enzyme I is autophosphorylated byphosphoenolpyruvate. Phosphorylated EI then catalyzes thephosphorylation of HPr in the membrane. HPr phosphorylates asugar-specific enzyme that is translocated across the membrane. Thus, E1and HPr are necessary for sugar translocation. The phosphotransferasesystem is reviewed by, for example, Postma et al. (1993, Microbiol.Rev., 57:543-94) and the pts operon is reviewed by, for example,Vadeboncoeur et al. (2000, J. Mol. Microbiol. Biotechnol., 2:483-90).

[0022] The invention provides methods to detect GAS by amplifying, forexample, GAS nucleic acid molecules corresponding to a portion of theptsI gene encoding enzyme I (EI) of the phosphoenolpyruvate:sugarphosphotransferase system. GAS nucleic acid molecules other than thoseexemplified herein (e.g., other than ptsI) also can be used to detectGAS in a sample and are known to those of skill in the art. Nucleic acidsequences encoding GAS ptsI have been described (see, for example,Ferretti et al., 2001, Proc. Natl. Acad. Sci. USA, 98:4658-63; andGenBank Accession Nos. NC 002737, and AE004092). Specifically, primersand probes to amplify and detect GAS ptsI nucleic acid molecules areprovided by the invention.

[0023] Primers that amplify a GAS nucleic acid molecule, e.g., a portionof the ptsI gene, can be designed using, for example, a computer programsuch as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.).Important features when designing oligonucleotides to be used asamplification primers include, but are not limited to, an appropriatesize amplification product to facilitate detection (e.g., byelectrophoresis), similar melting temperatures for the members of a pairof primers, and the length of each primer (i.e., the primers need to belong enough to anneal with sequence-specificity and to initiatesynthesis but not so long that fidelity is reduced duringoligonucleotide synthesis). Typically, oligonucleotide primers are 8 to50 nucleotides in length (e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).“ptsI primers” as used herein refers to oligonucleotide primers thatspecifically anneal to GAS nucleic acid sequences encoding ptsI andinitiate synthesis therefrom under appropriate conditions.

[0024] Designing oligonucleotides to be used as hybridization probes canbe performed in a manner similar to the design of primers, although themembers of a pair of probes preferably anneal to an amplificationproduct within no more than 5 nucleotides of each other on the samestrand such that fluorescent resonance energy transfer (FRET) can occur(e.g., within no more than 1, 2, 3, or 4 nucleotides of each other).This minimal degree of separation typically brings the respectivefluorescent moieties into sufficient proximity such that FRET occurs. Itis to be understood, however, that other separation distances (e.g., 6or more nucleotides) are possible provided the fluorescent moieties areappropriately positioned relative to each other (for example, with alinker arm) such that FRET can occur. In addition, probes can bedesigned to hybridize to targets that contain a mutation orpolymorphism, thereby allowing differential detection of GAS strainsbased on either absolute hybridization of different pairs of probescorresponding to the particular GAS strain to be distinguished ordifferential melting temperatures between, for example, members of apair of probes and each amplification product corresponding to a GASstrain to be distinguished. For example, using appropriate probe pairs,group A streptococcus (S. pyogenes) can be distinguished from otherstreptococcal strains (for example, group B streptococcus (S.agalactiae), group C streptococcus (e.g., S. equisimillis) and group Gstreptococcus (e.g., S. canis)). As with oligonucleotide primers,oligonucleotide probes usually have similar melting temperatures, andthe length of each probe must be sufficient for sequence-specifichybridization to occur but not so long that fidelity is reduced duringsynthesis. Oligonucleotide probes are 8 to 50 nucleotides in length(e.g., 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, or 50 nucleotides in length). “ptsI probes” as usedherein refers to oligonucleotide probes that specifically anneal to aptsI amplification product.

[0025] Constructs of the invention include vectors containing a GASnucleic acid molecule, e.g., a GAS ptsI gene or fragment thereof.Constructs can be used, for example, as a control template nucleic acid.Vectors suitable for use in the present invention are commerciallyavailable and/or produced by recombinant DNA technology methods routinein the art. A GAS ptsI nucleic acid molecule can be obtained, forexample, by chemical synthesis, direct cloning from GAS, or by PCRamplification. A GAS nucleic acid molecule or fragments thereof can beoperably linked to a promoter or other regulatory element such as anenhancer sequence, a response element or an inducible element thatmodulates expression of the GAS nucleic acid molecule. As used herein,operably linking refers to connecting a promoter and/or other regulatoryelements to a GAS nucleic acid molecule in such a way as to permitand/or regulate expression of the GAS nucleic acid molecule. A promoterthat does not normally direct expression of GAS ptsI can be used todirect transcription of a ptsI nucleic acid molecule using, for examplea viral polymerase, a bacterial polymerase, or a eukaryotic RNApolymerase I. Alternatively, the ptsI native promoter can be used todirect transcription of a ptsI nucleic acid molecule using, for example,an S. pyogenes RNA polymerase or a host RNA polymerase. In addition,operably linked can refer to an appropriate connection between a GASptsI promoter or other regulatory element to a heterologous codingsequence (i.e., a non-ptsI coding sequence, for example a reporter gene)in such a way as to permit expression of the heterologous codingsequence.

[0026] Constructs suitable for use in the methods of the inventiontypically include, in addition to a GAS ptsI nucleic acid molecule,sequences encoding a selectable marker (e.g., an antibiotic resistancegene) for selecting desired constructs and/or transformants, and anorigin of replication. The choice of vector systems usually depends uponseveral factors, including, but not limited to, the choice of hostcells, replication efficiency, selectability, inducibility, and the easeof recovery.

[0027] Constructs of the invention containing a GAS ptsI nucleic acidmolecule can be propagated in a host cell. As used herein, the term hostcell is meant to include prokaryotes and eukaryotes such as yeast, plantand animal cells. Prokaryotic hosts can include E. coli, Salmonellatyphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hostsinclude yeasts such as S. cerevisiae, S. pombe, and Pichia pastoris,mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells,insect cells, and plant cells such as Arabidopsis thaliana and Nicotianatabacurn. A construct of the invention can be introduced into a hostcell using any of the techniques commonly known to those of ordinaryskill in the art. For example, calcium phosphate precipitation,electroporation, heat shock, lipofection, microinjection, andviral-mediated nucleic acid transfer are common methods for introducingnucleic acids into host cells. In addition, naked DNA can be delivereddirectly to cells (see, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466).

[0028] Polymerase Chain Reaction (PCR)

[0029] U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188disclose conventional PCR techniques. PCR typically employs twooligonucleotide primers that bind to a selected nucleic acid template(e.g., DNA or RNA). Primers useful in the present invention includeoligonucleotides capable of acting as a point of initiation of nucleicacid synthesis within a GAS ptsI nucleic acid sequence. A primer can bepurified from a restriction digest by conventional methods, or it can beproduced synthetically. A primer is preferably single-stranded formaximum efficiency in amplification, but a primer can bedouble-stranded. Double-stranded primers are first denatured, i.e.,treated to separate the strands. One method of denaturing doublestranded nucleic acids is by heating.

[0030] The term “thermostable polymerase” refers to a polymerase enzymethat is heat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3 ′ direction along the templatestrand. Thermostable polymerases have been isolated from Thermus flavus,T. ruber, T thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCRprovided the enzyme is replenished.

[0031] If the GAS template nucleic acid is double-stranded, it isnecessary to separate the two strands before it can be used as atemplate in PCR. Strand separation can be accomplished by any suitabledenaturing method including physical, chemical or enzymatic means. Onemethod of separating the nucleic acid strands involves heating thenucleic acid until it is predominately denatured (e.g., greater than50%, 60%, 70%, 80%, 90% or 95% denatured). The heating conditionsnecessary for denaturing template nucleic acid will depend, e.g., on thebuffer salt concentration and the length and nucleotide composition ofthe nucleic acids being denatured, but typically range from about 90° C.to about 105° C. for a time depending on features of the reaction suchas temperature and the nucleic acid length. Denaturation is typicallyperformed for about 0 sec to 4 min.

[0032] If the double-stranded nucleic acid is denatured by heat, thereaction mixture is allowed to cool to a temperature that promotesannealing of each primer to its target sequence on the GAS nucleic acid.The temperature for annealing is usually from about 35° C. to about 65°C. The reaction mixture is then adjusted to a temperature at which theactivity of the polymerase is promoted or optimized, e.g., a temperaturesufficient for extension to occur from the annealed primer to generateproducts complementary to the template nucleic acid. The temperatureshould be sufficient to synthesize an extension product from each primerthat is annealed to a nucleic acid template, but should not be so highas to denature an extension product from its complementary template. Thetemperature generally ranges from about 40° to 80° C.

[0033] PCR assays can employ GAS nucleic acid such as DNA or RNA,including messenger RNA (mRNA). The template nucleic acid need not bepurified; it may be a minor fraction of a complex mixture, such as GASnucleic acid contained in human cells. DNA or RNA may be extracted fromany biological sample such as a throat swab, tissue (e.g., skin, orlymph node) or body fluids (e.g., cerebrospinal fluid (CSF), blood, orurine) by routine techniques such as those described in DiagnosticMolecular Microbiology: Principles and Applications (Persing et al.(eds), 1993, American Society for Microbiology, Washington D.C).Template nucleic acids can be obtained from any number of sources, suchas plasmids, or natural sources including bacteria, yeast, viruses,organelles, or higher organisms such as plants or animals.

[0034] The oligonucleotide primers are combined with other PCR reagentsunder reaction conditions that induce primer extension. For example,chain extension reactions generally include50 mM KCl, 10 mM Tris-HCl (pH8.3), 1.5 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 μg denatured templateDNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase,and 10% DMSO. The reactions usually contain 150 to 320 μM each of dATP,dCTP, dTTP, dGTP, or one or more analogs thereof.

[0035] The newly synthesized strands form a double-stranded moleculethat can be used in the succeeding steps of the reaction. The steps ofstrand separation, annealing, and elongation can be repeated as often asneeded to produce the desired quantity amplification productscorresponding to the target GAS nucleic acid molecule. The limitingfactors in the reaction are the amounts of primers, thermostable enzyme,and nucleoside triphosphates present in the reaction. The cycling steps(i.e., amplification and hybridization) are preferably repeated at leastonce. For use in detection, the number of cycling steps will depend,e.g., on the nature of the sample. If the sample is a complex mixture ofnucleic acids, more cycling steps may be required to amplify the targetsequence sufficient for detection. Generally, the cycling steps arerepeated at least about 20 times, but may be repeated as many as 40, 60,or even 100 times.

[0036] Fluorescent Resonance Energy Transfer (FRET)

[0037] FRET technology (see, for example, U.S. Pat. Nos. 4,996,143,5,565,322, 5,849,489, and 6,162,603) is based on the fact that when adonor and a corresponding acceptor fluorescent moiety are positionedwithin a certain distance of each other, energy transfer takes placebetween the two fluorescent moieties that can be visualized or otherwisedetected and/or quantitated. As used herein, two oligonucleotide probes,each containing a fluorescent moiety, can hybridize to an amplificationproduct at particular positions determined by the complementarity of theoligonucleotide probes to the GAS target nucleic acid sequence. Uponhybridization of the oligonucleotide probes to the amplification productat the appropriate positions, a FRET signal is generated.

[0038] Fluorescent analysis can be carried out using, for example, aphoton counting epifluorescent microscope system (containing theappropriate dichroic mirror and filters for monitoring fluorescentemission at the particular range), a photon counting photomultipliersystem or a fluorometer. Excitation to initiate energy transfer can becarried out with an argon ion laser, a high intensity mercury (Hg) arclamp, a fiber optic light source, or other high intensity light sourceappropriately filtered for excitation in the desired range.

[0039] As used herein with respect to donor and corresponding acceptorfluorescent moieties, “corresponding” refers to an acceptor fluorescentmoiety having an emission spectrum that overlaps the excitation spectrumof the donor fluorescent moiety. The wavelength maximum of the emissionspectrum of the acceptor fluorescent moiety preferably should be atleast 100 nm greater than the wavelength maximum of the excitationspectrum of the donor fluorescent moiety. Accordingly, efficientnon-radiative energy transfer can be produced therebetween.

[0040] Fluorescent donor and corresponding acceptor moieties aregenerally chosen for (a) high efficiency Förster energy transfer; (b) alarge final Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

[0041] Representative donor fluorescent moieties that can be used withvarious acceptor fluorescent moieties in FRET technology includefluorescein, Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate,Lucifer Yellow VS,4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC™-Red 640, LC™-Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0042] The donor and acceptor fluorescent moieties can be attached tothe appropriate probe oligonucleotide via a linker arm. The length ofeach linker arm can be important, as the linker arms will affect thedistance between the donor and the acceptor fluorescent moieties. Thelength of a linker arm for the purpose of the present invention is thedistance in Angstroms (Å) from the nucleotide base to the fluorescentmoiety. In general, a linker arm is from about 10 to about 25 Å. Thelinker arm may be of the kind described in WO 84/03285. WO 84/03285 alsodiscloses methods for attaching linker arms to particular nucleotidebases, and also for attaching fluorescent moieties to a linker arm.

[0043] An acceptor fluorescent moiety such as an LC™-Red 640-NHS-estercan be combined with C6-Phosphoramidites (available from ABI (FosterCity, Calif.) or Glen Research (Sterling, Va.)) to produce, for example,LC™-Red 640-Phosphoramidite. Frequently used linkers to couple a donorfluorescent moiety such as fluorescein to an oligonucleotide includethiourea linkers (FITC-derived, for example, fluorescein-CPG's from GlenResearch or ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPG's that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

[0044] Detection of Group A Streptococcus (GAS)

[0045] Cell culture is considered the gold standard for detection ofGAS. Culture, however, suffers from slow turnaround time (e.g., 1 to 2days). There are a number of variations on the methodology of cellculture that are used for the detection of GAS. Culture of throat swabsis generally done by streaking a patient's swab on a plate containing,for example, T-soy blood agar. Following incubation, GAS is identifiedby the presence of β-hemolytic colonies. Culture is usually used inconjunction with an antigen test to confirm the presence of GAS. Forexample, β-hemolytic colonies can be tested for the presence of thegroup A antigen using a fluorescently labeled antibody, or a bacitracindisk can be placed on the plate. Bacitracin inhibits the growth ofβ-hemolytic colonies.

[0046] There are a number of rapid antigen tests on the market that useantibodies directed against, for example, the group A antigen. Ananalysis of the rapid antigen tests performed by nurses andtechnologists found sensitivities in the 50 to 60% range. A laboratorycomparision using TestPack® Plus™ Strep A (Abbott Laboratories, AbbottPark, Ill.) found 68% sensitivity. The rapid antigen assays currentlyavailable are not sensitive enough to replace culture, i.e., to serve asstand-alone diagnostic assays, and the Infectious Disease Society ofAmerica (IDSA) has recommended that rapid antigen tests for S. pyogenesbe backed up with culture.

[0047] Over the past 15 years, a number of rapid test formats,frequently referred to as “rapid strep screens” (RSSs), have emerged.Generally, these assays are modifications of the immunoassay methods andinclude simple, single-use devices adapted for manual use. Single-useimmunoassay devices are classified as moderately complex under theClinical Laboratory Improvement Amendment (CLIA, 1988) guidelines.Because they can be performed quickly, relatively inexpensively, andrequire few additional reagents, they are suited to a variety ofphysician office testing environments. Latex agglutination is awell-established immunoassay method in which latex particles are coatedwith an analyte-specific capture reagent, such as an antibody. The majorlimitations of agglutination-based assays are their lack of sensitivityand specificity and the subjective nature of test result interpretation.However, because these tests are fast, inexpensive, and require minimalreagents, they have been widely used. Other variations of immunoassaytechnology are the flow-through membrane devices. Hybritech's® (SanDiego, Calif.) ICON® format is based on this method and is available forthe detection of a number of infectious diseases. Other flow-throughmembrane tests include Kodak's (Rochester, N.Y.) SURECELL® and BectonDickinson's (Franklin Lakes, N.J.) Qtest®. Rapid assays such as theDirectigen 1-2-3 (Becton Dickinson & Co., Sparks, Md.) can alsoincorporate liposomes.

[0048] A second-generation immunoassay technology is available inBioStar's® (Boulder, Colo.) Optical ImmunoAssay (OIA®) test. In thistest, a solid reflective support is coated with thin film selected tospecifically attenuate the reflection of certain wavelengths of visiblelight through destructive interference, thereby producing the device'scharacteristic gold background color. Any change in the mass on thesurface of the device due to analyte binding modifies the thin film andshifts the attenuated wavelengths, resulting in a color change from goldto purple. The OIA test requires a very small sample such that repeatedor additional testing can be performed without collecting multiplespecimens from the patient. Strep A OIA assays are significantly moresensitive than first-generation rapid strep screens and studies thatexamined the Strep A OIA® assay compared with culture found 81-92%sensitivity of the Strep A OIA® assay. As with other rapid assays, theOIA method has been classified as moderately complex.

[0049] The Group A Streptococcus Direct (GASD) is a commerciallyavailable assay that uses nucleic acid hybridization for the qualitativedetection of GAS RNA. See GenProbe Inc., San Diego, Calif.;http:H/www.gen-probe.com/gasd.html. GenProbe reports that the assay hasa sensitivity of 91.7% and a specificity of 99.3%. Other studies haveshown the GASD to be 86% sensitive when compared to a 72-hour cellculture assay and 93% sensitive when compared to standard culturemethods that include serotyping of colonies. For most diagnostic orclinical laboratories, this level of sensitivity is not high enough toallow GASD to replace culture.

[0050] The real-time assay described herein has been compared to cellculture and to a rapid antigen test using 500 patient specimens. Thereal-time PCR method is more sensitive than culture and far superior insensitivity to the rapid antigen test. The specificity was alsodetermined using DNA from cultures of a variety of streptococcal andnon-streptococcal microorganisms commonly found in the throat andrespiratory tract.

[0051] The invention provides methods for detecting the presence orabsence of GAS in a biological sample from an individual. Methodsprovided by the invention avoid problems of sample contamination, falsenegatives and false positives. The methods include performing at leastone cycling step that includes amplifying and hybridizing. Anamplification step includes contacting the biological sample with a pairof ptsI-primers to produce a ptsI amplification product if a GAS ptsInucleic acid molecule is present in the sample. Each of the ptsI primersanneals to a target within or adjacent to a GAS ptsI nucleic acidmolecule such that at least a portion of the amplification productcontains nucleic acid sequence corresponding to ptsI and, moreimportantly, such that the amplification product contains the nucleicacid sequences that are complementary to ptsI probes. A hybridizing stepincludes contacting the sample with a pair of ptsI probes. Generally,the members of the pair of ptsI probes hybridize to the amplificationproduct within no more than five nucleotides of each other. According tothe invention, a first ptsI probe of the pair of ptsI probes is labeledwith a donor fluorescent moiety and a second ptsI probe of the pair ofptsI probes is labeled with a corresponding acceptor fluorescent moiety.The method further includes detecting the presence of absence of FRETbetween the donor fluorescent moiety of the first ptsI probe and thecorresponding acceptor fluorescent moiety of the second ptsI probe.Multiple cycling steps can be performed, preferably in a thermocycler.The above-described methods for detecting GAS in a biological sampleusing primers and probes directed toward ptsI also can be performedusing other GAS gene-specific primers and probes.

[0052] As used herein, “amplifying” refers to the process ofsynthesizing nucleic acid molecules that are complementary to one orboth strands of a template nucleic acid (e.g., ptsI GAS nucleic acidmolecules). Amplifying a nucleic acid molecule typically includesdenaturing the template nucleic acid, annealing primers to the templatenucleic acid at a temperature that is below the melting temperatures ofthe primers, and enzymatically elongating from the primers to generatean amplification product. The denaturing, annealing and elongating stepseach can be performed once. Generally, however, the denaturing,annealing and elongating steps are performed multiple times such thatthe amount of amplification product is increasing, oftentimesexponentially, although exponential amplification is not required by thepresent methods. Amplification typically requires the presence ofdeoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g.,Platinum® Taq) and an appropriate buffer and/or co-factors for optimalactivity of the polymerase enzyme (e.g., MgCl₂ and/or KCl).

[0053] If amplification of GAS nucleic acid occurs and an amplificationproduct is produced, the step of hybridizing results in a detectablesignal based upon FRET between the members of the pair of probes. Asused herein, “hybridizing” refers to the annealing of probes to anamplification product. Hybridization conditions typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

[0054] Generally, the presence of FRET indicates the presence of GAS inthe biological sample, and the absence of FRET indicates the absence ofGAS in the biological sample. Inadequate specimen collection,transportation delays, inappropriate transportation conditions, or useof certain collection swabs (e.g., calcium alginate or aluminum shaft)are all conditions that can affect the success and/or accuracy of thetest result, however. Using the methods disclosed herein, detection ofFRET within 40 cycling steps is indicative of a GAS infection.

[0055] Representative biological samples that can be used in practicingthe methods of the invention include throat swabs, tissues, or bodilyfluids. Biological sample collection and storage methods are known tothose of skill in the art. Biological samples can be processed (e.g., bystandard nucleic acid extraction methods and/or using commercial kits)to release GAS nucleic acid or, in some cases, the biological sample iscontacted directly with the PCR reaction components and the appropriateoligonucleotides.

[0056] Melting curve analysis is an additional step that can be includedin a cycling profile. Melting curve analysis is based on the fact thatDNA melts at a characteristic temperature called the melting temperature(Tm), which is defined as the temperature at which half of the DNAduplexes have separated into single strands. The melting temperature ofa DNA depends primarily upon its nucleotide composition. Thus, DNAmolecules rich in G and C nucleotides have a higher Tm than those havingan abundance of A and T nucleotides. By detecting the temperature atwhich signal is lost, the melting temperature of probes can bedetermined. Similarly, by detecting the temperature at which signal isgenerated, the annealing temperature of probes can be determined. Themelting temperature(s) of the ptsI probes from the ptsI amplificationproduct can confirm the presence of GAS in the sample.

[0057] Within each thermocycler run, control samples can be cycled aswell. Positive control samples can amplify control nucleic acid template(e.g., template other than ptsI) using, for example, control primers andcontrol probes. Positive control samples can also amplify, for example,a plasmid construct containing GAS ptsI nucleic acid molecules. Such aplasmid control can be amplified internally (e.g., within eachbiological sample) or in separate samples run side-by-side with thepatients' samples. Each thermocycler run also should include a negativecontrol that, for example, lacks GAS template DNA. Such controls areindicators of the success or failure of the amplification,hybridization, and/or FRET reaction. Therefore, control reactions canreadily determine, for example, the ability of primers to anneal withsequence-specificity and to initiate elongation, as well as the abilityof probes to hybridize with sequence-specificity and for FRET to occur.

[0058] In an embodiment, the methods of the invention include steps toavoid contamination. For example, an enzymatic method utilizinguracil-DNA glycosylase is described in U.S. Pat. Nos. 5,035,996,5,683,896 and 5,945,313 to reduce or eliminate contamination between onethermocycler run and the next. In addition, standard laboratorycontainment practices and procedures are desirable when performingmethods of the invention. Containment practices and procedures include,but are not limited to, separate work areas for different steps of amethod, containment hoods, barrier filter pipette tips and dedicated airdisplacement pipettes. Consistent containment practices and proceduresby personnel are desirable for accuracy in a diagnostic laboratoryhandling clinical samples.

[0059] Conventional PCR methods in conjunction with FRET technology canbe used to practice the methods of the invention. In one embodiment, aLightCycler™ instrument is used. A detailed description of theLightCycler™ System and real-time and on-line monitoring of PCR can befound at http://biochem.roche.com/lightcycler. The following patentapplications describe real-time PCR as used in the LightCycler™technology: WO 97/46707, WO 97/46714 and WO 97/46712. The LightCycler™instrument is a rapid thermocycler combined with a microvolumefluorometer utilizing high quality optics. This rapid thermocyclingtechnique uses thin glass cuvettes as reaction vessels. Heating andcooling of the reaction chamber are controlled by alternating heated andambient air. Due to the low mass, of air and the high ratio of surfacearea to volume of the cuvettes, very rapid temperature exchange ratescan be achieved within the LightCycler™ thermal chamber. Addition ofselected fluorescent dyes to the reaction components allows the PCR tobe monitored in real-time and on-line. Furthermore, the cuvettes serveas an optical element for signal collection (similar to glass fiberoptics), concentrating the signal at the tip of the cuvettes. The effectis efficient illumination and fluorescent monitoring of microvolumesamples.

[0060] The LightCycler™ carousel that houses the cuvettes can be removedfrom the instrument. Therefore, samples can be loaded outside of theinstrument (in a PCR Clean Room, for example). In addition, this featureallows for the sample carousel to be easily cleaned and sterilized. Thefluorometer, as part of the LightCycler™ apparatus, houses the lightsource. The emitted light is filtered and focused by an epi-illuminationlens onto the top of the cuvettes. Fluorescent light emitted from thesample is then focused by the same lens, passed through a dichroicmirror, filtered appropriately, and focused onto data-collectingphotohybrids. The optical unit currently available in the LightCycler™instrument (Catalog No. 2 011 468) includes three band-pass filters (530nm, 640 nm, and 710 nm), providing three-color detection and severalfluorescence acquisition options. Data collection options include onceper cycling step monitoring, fully continuous single-sample acquisitionfor melting curve analysis, continuous sampling (in which samplingfrequency is dependent on sample number) and/or stepwise measurement ofall samples after defined temperature interval.

[0061] The LightCycler™ can be operated using a PC workstation and canutilize a Windows NT operating system. Signals from the samples areobtained as the machine positions the capillaries sequentially over theoptical unit. The software can display the fluorescence signals inreal-time immediately after each measurement. Fluorescent acquisitiontime is 10-100 msec. After each cycling step, a quantitative display offluorescence vs. cycle number can be continually updated for allsamples. The data generated can be stored for further analysis.

[0062] A common FRET technology format utilizes two hybridizationprobes. Each probe can be labeled with a different fluorescent moietyand the two probes are generally designed to hybridize in closeproximity to each other in a target DNA molecule (e.g., an amplificationproduct). By way of example, a donor fluorescent moiety such asfluorescein can be excited at 470 nm by the light source of theLightCycler™ Instrument. During FRET, fluorescein transfers its energyto an acceptor fluorescent moiety such as LightCycler™-Red 640 (LC™-Red640) or LightCycler™-Red 705 (LC™-Red 705). The acceptor fluorescentmoiety then emits light of a longer wavelength (e.g., 640 nm or 705 nm,respectively), which is detected by the optical detection system of theLightCycler™ instrument. Other donor and corresponding acceptorfluorescent moieties suitable for use in the invention are describedabove. Efficient FRET can only take place when the fluorescent moietiesare in direct local proximity (for example, within 5 nucleotides of eachother as described above) and when the emission spectrum of the donorfluorescent moiety overlaps with the absorption spectrum of the acceptorfluorescent moiety. The intensity of the emitted signal can becorrelated with the number of original target DNA molecules (e.g., thenumber of GAS organisms).

[0063] Another FRET technology format utilizes TaqMan® technology todetect the presence or absence of an amplification product, and hence,the presence or absence of GAS. TaqMan® technology utilizes onesingle-stranded hybridization probe labeled with two fluorescentmoieties. When a first fluorescent moiety is excited with light of asuitable wavelength, the absorbed energy is transferred to a secondfluorescent moiety according to the principles of FRET. The secondfluorescent moiety is generally a quencher molecule. During theannealing step of the PCR reaction, the labeled hybridization probebinds to the target DNA (i.e., the amplification product) and isdegraded by the 5′ to 3′ exonuclease activity of the Taq Polymeraseduring the subsequent elongation phase. As a result, the excitedfluorescent moiety and the quencher moiety become spatially separatedfrom one another. As a consequence, upon excitation of the firstfluorescent moiety in the absence of the quencher, the fluorescenceemission from the first fluorescent moiety can be detected. By way ofexample, an ABI PRISM® 7700 Sequence Detection System (AppliedBiosystems, Foster City, Calif.) uses TaqMan® technology, and issuitable for performing the methods described herein for detecting GAS.Information on PCR amplification and detection using an ABI PRISM® 770system can be found at http://www.appliedbiosystems.com/products.

[0064] Yet another FRET technology format utilizes molecular beacontechnology to detect the presence or absence of an amplificationproduct, and hence, the presence or absence of GAS. Molecular beacontechnology uses a hybridization probe labeled with a donor fluorescentmoiety and an acceptor fluorescent moiety. The acceptor fluorescentmoiety is generally a quencher, and the fluorescent labels are typicallylocated at each end of the probe. Molecular beacon technology uses aprobe oligonucleotide having sequences that permit secondary structureformation (e.g., a hairpin). As a result of secondary structureformation within the probe, both fluorescent moieties are in spatialproximity when the probe is in solution. After hybridization to thetarget nucleic acids (i.e., the amplification products), the secondarystructure of the probe is disrupted and the fluorescent moieties becomeseparated from one another such that after excitation with light of asuitable wavelength, the emission of the first fluorescent moiety can bedetected.

[0065] As an alternative to detection using FRET technology, anamplification product can be detected using a nucleic acid binding dyesuch as a fluorescent DNA binding dye (e.g., SYBRGreenI® or SYBRGold®(Molecular Probes)). Upon interaction with the double-stranded nucleicacid, such nucleic acid binding dyes emit a fluorescence signal afterexcitation with light at a suitable wavelength. A nucleic acid bindingdye such as a nucleic acid intercalating dye also can be used. Whennucleic acid binding dyes are used, a melting curve analysis is usuallyperformed for confirmation of the presence of the amplification product.

[0066] It is understood that the present invention is not limited by theconfiguration of one or more commercially available instruments.

[0067] Articles of Manufacture

[0068] The invention further provides for articles of manufacture todetect GAS. An article of manufacture according to the present inventioncan include primers and probes used to detect GAS, together withsuitable packaging material. Representative primers and probes providedin a kit for detection of GAS can be complementary to GAS ptsI nucleicacid molecules. Methods of designing primers and probes are disclosedherein, and representative examples of primers and probes that amplifyand hybridize to GAS ptsI nucleic acid molecules are provided.

[0069] Articles of manufacture of the invention also can include one ormore fluorescent moieties for labeling the probes or, alternatively, theprobes supplied with the kit can be labeled. For example, an article ofmanufacture may include a donor fluorescent moiety for labeling one ofthe ptsI probes and a corresponding acceptor fluorescent moiety forlabeling the other ptsI probe. Examples of suitable FRET donorfluorescent moieties and corresponding acceptor fluorescent moieties areprovided herein.

[0070] Articles of manufacture of the invention also can contain apackage insert having instructions thereon for using pairs of ptsIprimers and ptsI probes to detect GAS in a biological sample. Articlesof manufacture may additionally include reagents for carrying out themethods disclosed herein (e.g., buffers, polymerase enzymes, co-factors,or agents to prevent contamination). Such reagents may be specific forone of the commercially available instruments described herein.

[0071] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1

[0072] Sample Preparation

[0073] The end of a Culturette (Becton Dickinson Microbiology Systems,4360210) was wetted with Stuart's transport medium and placed in a 0.65ml tube of a Swab Extraction Tube System (SETS). A SETS is prepared byusing a 2 ml screw capped centrifuge tubes (Sarstedt 72.693.005) and a0.65 ml centrifuge tube (Intermountain Scientific Corporation,C-3300-2). An awl was used to puncture a hole in the bottom of the 0.65ml centrifuge tube, which is nested inside the 2 ml tube. The handle ofthe swab was covered with a Bio-Screen® Biohazard wipe (Fisher14-412-52C), broken off near the top of the tube and the lid was closed.The SETS was centrifuged at 20,800×g for 3 min in an Eppendorf 5741C.The 0.65 ml tube from the SETS was discarded. The supernatant wascarefully removed and discarded with a fine-tip transfer pipet. Onehundred μl water was added to the pellet, the tube was capped and placedin-a 100° C. heating block for 10 min. The tube was then centrifuged at20,800×g for 3 min. The LightCycler cuvettes were placed in theLightCycler rotor and 15 μl PCR mix was placed in each tube. Five μl ofsample supernatant was added to the 15 μl of PCR mix in the cuvette andthe cuvette was capped. The LightCycler cuvette was centrifuged at1000×g for 30 sec and placed in the LightCycler apparatus.

[0074] A colony from a blood agar plate of Streptococcus pyogenes (ATCC19615) was inoculated into 5 ml of Todd-Hewitt broth and incubatedovernight at 37° C. for use as a positive control. The turbidity of theculture was adjusted to a McFarland 0.5 standard. A 0.5 ml sample of theculture was placed in a 2 ml screw capped tube and placed in a 100° C.heating block for 10 min. The culture was diluted 1/1000 with water andstored at 4° C. In some cases, a plasmid containing the cloned ptsIamplification product was diluted to 100 copies per μl and used as apositive control.

Example 2

[0075] Primers and Probes

[0076] ptsI primers were synthesized by the Mayo Core Facility on a 0.2nm scale, and were quantitated by UV absorption at 260 nm and mixedtogether to make a solution containing 25 μM of each primer.

[0077] Probes were synthesized by IT Biochem, and were dissolved in TE′to a final concentration of 20 μM (supplied with the probes andresuspended according to manufacturer's instructions). The concentrationof oligonucleotides and dye was double-checked by UV absorption usingthe following equations (Biochemica 1:5-8, 1999):$\lbrack{dye}\rbrack = {{\frac{A_{dye}}{E_{dye}}\quad\lbrack{oligo}\rbrack} = \frac{A_{260} - \left( {A_{260} \times \frac{E_{260{({dye})}}}{E_{dye}}} \right)}{\frac{10^{6}}{{nmol}/A_{260}}}}$

[0078] To determine the natural sequence variation in the ptsI gene, theDNA sequence was determined for 11 isolates of group A streptococcusfrom the Mayo Foundation culture collection. The sequences obtained werealigned to the ptsI gene from the M1 strain of group A Streptococcus atthe University of Oklahoma (Ferretti et al., 2001, Proc. Natl. Acad.Sci. USA, 98:4658-63; GenBank Accession No. AE004092). The ptsI targetsequence between base pairs 170 and 1543 was found to be mostlyconserved among isolates of group A streptococcus (FIG. 1). Most of thepolymorphisms found were silent mutations in the third base pair of thecodon. The sequence variation of the ptsI gene from a number of otherstreptococcus species that can be found in an oral-pharyngeal sample wasdetermined using primers designed to conserved regions of the ptsI gene.

[0079] From these alignments, primers and probes directed toward ptsIwere designed. The positions of the ptsI primers were 180 to 200 (ptsU)and 357 to 377 (ptsL) of Ferreti et al., resulting in a 198 bp PCRproduct. The ptsI probe positions are 242 to 260 for thefluorescein-labeled probe and 262 to 285 for the Red-640-labeled proberelative to Ferreti et at. The ptsF3 probe was provided already labeledon its 3′ end with fluorescein and the ptsR1 probe was provided alreadylabeled on its 5′ end with Red-640.

[0080] The sequences of the ptsI primers are: ptsU: 5′-AAA TGC AGT AGAAAG CTT AGG-3′; and ptsL: 5′-TGC ATG TAT GGG TTA TCT TCC-3′.

[0081] The sequences of the ptsI probes are: ptsF3: 5′-TTG CTG ATC CAGAAA TGA T-3′; and ptsR1: 5′-AGC CAG GTT AAA GAA ACG ATT CGC-3′.

Example 3

[0082] Conditions for Real-time PCR

[0083] Reaction mixtures for detecting GAS using ptsI primers and probeswere made according to the following chart. Group A Strep Detectionreagent 10X Volume Final Stock Stock (μl) Conc. Water — —  45 — Mg* 200mM 30 mM  15 3 mM Primer ptsU 50 μM 5 μM  10 μM Primer ptsL 50 μM 5 μM 10 0.5 μM Probe ptsF3 20 μM 2 μM  10 μM Probe ptsR1 20 μM 2 μM  10 0.2μM Total — — 100 —

[0084] Each 15 μl of group A Strep PCR mix contains 2 μl Group A StrepDetection reagent, 2 μl LightCycler-FastStart DNA Master HybridizationProbes Reagent (Roche 3 003 248) and 11 μl water.

[0085] Conditions for the real-time PCR using the LightCycler instrumentto detect GAS in biological samples were as follows. The gains were setat 1, 5, and 15 for channels F1, F2, and F3, respectively. Program TempName/ Transition Analysis Analysis Temp Time Rate Signal mode modeCycles (° C.) (sec) (° C./sec) Acquisition Denature None 1 95 600 20None PCR Quantif. 40 95 10 20 None 55 10 20 Single 72 8 20 None MeltMelt 1 95 0 20 None Analysis 45 10 2 None 70 0 0.2 Continuous Cool None1 35 0 20 None

Example 4

[0086] Reporting Results

[0087] Analysis of the real-time PCR data is shown in FIG. 3. The meltanalysis is shown in FIG. 4 and confirms the amplification productidentification as group A streptococcus with melting temperatures withintwo degrees of the positive control, which is typically 56° C. to 58° C.Groups C and G streptococcus produce a melting peak of 50° C. to 52° C.,but are not detected during the quantification portion of the real timePCR due to the presence of nucleotide polymorphisms at the probe bindingsites. Thus, a reaction -was reported as positive for Group AStreptococcus if a positive quantification signal with a melting curvesimilar to the positive control was observed. All other reactions werereported as negative.

Example 5

[0088] Validation Studies

[0089] Specificity of the method was determined by performing real-timePCR as described above on streptococcus and non-streptococcus organisms.DNA from the following organisms was tested using the ptsI primers andprobes and none tested positive based on FRET detection. RespiratoryPanel S. aureus (ATCC 29213 S epidermidis human DNA E. coli Ps.aeruginosa K. pneumophilia H influenza Aeromonas spp L. jordanis S.maltophilia K. oxytoca P. cepacia P. fluorescens P. mirabilisAcinetobacter spp Morganella spp. P. vulgaris M. pneumonia C. jeluni M.catarrhalis C. pneumonia L. monocytogenes L. pneumophila B.bronchioseptica B. holmesii B. pertussis B. parapertussis

[0090] Strep Group: S. suis S. viridans L. latis S. anginosus S. equi S.uberis S. MG-intermedius S. mutans E. faecium S. bovis E. faecalis S.mitis S. dysgalactiae S. canis S. salivarius S. eguinus S. pneumococcusGroup F Strep Group B Strep non-beta Group C strep Group G strep

[0091] Using plasmid-derived ptsI nucleic acid, the analyticalsensitivity was determined to be less than 20 copies of target nucleicacid per reaction.

[0092] Sensitivity was also determined by testing dilutions of asuspension of S. pyogenes (ATCC 19615) grown in broth overnight. Thecolony forming units (cfu) per ml was determined by spread platingdilutions of the culture. The sensitivity was determined to be 0.13cfu/ml. This sensitivity is acceptable since S. pyogenes grows in chainsof 5-15 cells, and each chain of cells is only counted as one cfu. Thus,a cfu would be expected to contain at least 5-15 copies of the target.

Example 6

[0093] Comparison of Methods

[0094] The LightCycler assay for GAS using a ptsI target was compared toconventional culture (with samples cultured at the Mayo MicrobiologyLaboratory) and a rapid antigen test, Directigen 1-2-3™ Group A StrepTest, from Becton Dickinson. Cultures were performed on Strep SelectiveAgar and positives were identified using a fluorescent antibody stain(BBL) that allows for detection of low numbers of GAS. Double swabthroat swabs were used to collect patient specimens. One throat swab wasused in the conventional culture and rapid antigen test procedure. Theother swab was treated to extract the DNA and analyzed using theLightCycler assay with the ptsI primers and probes.

[0095] The culture method is usually considered to be the “goldstandard” for detecting GAS from throat swabs. The results of each assayalso can be compared to all positive results. Such a method provides astandard to compare all the assays. The sensitivities of the LightCyclerassay using the ptsI target is more sensitive than culture and muchbetter than the rapid antigen test for detecting GAS. Culture Rapid Ag:Positive Negative Totals Positive 29  1  30 Negative 22 311 333 Totals51 312 363

[0096] Culture ptsI LC: Positive Negative Totals Positive 48  6  54Negative  3 306 309 Totals 51 312 363

[0097] Other Embodiments

[0098] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1 17 1 21 DNA Artificial Sequence Oligonucleotide 1 aaatgcagtagaaagcttag g 21 2 21 DNA Artificial Sequence Oligonucleotide 2tgcatgtatg ggttatcttc c 21 3 19 DNA Artificial Sequence Oligonucleotide3 ttgctgatcc agaaatgat 19 4 24 DNA Artificial Sequence Oligonucleotide 4agccaggtta aagaaacgat tcgc 24 5 1803 DNA Group A Streptococcus ptsIsequence from Oklahoma University M1 strain 5 atgacagaaa tgcttaaaggaattgcagcc tcagacggcg ttgctgttgc taaagcatat 60 ctactagttc agccggatttgtcatttgag actgttacag tcgcagatac aaatgcagaa 120 gaagctcgcc ttgatgttgcactccaagct gcacaagacg agctttctgt tatccgtgaa 180 aatgcagtag aaagcttaggtgaagaagca gcagccgttt ttgatgccca tttgatggtt 240 cttgctgatc cagaaatgatcagccaggtt aaagaaacga ttcgcgcaaa acaaacgaat 300 gcagaaacag gtcttaaagaagtgactgac atgttcatca ccatctttga aggcatggaa 360 gataacccat acatgcaagaacgtgcagcg gacatccgcg acgttgcaaa acgtgtgttg 420 gctcaccttt taggtgtaaaacttccaaat ccagctacaa tcaatgaaga atcaatcgtt 480 atcgcacacg atttgacaccttcagatact gctcaactta acaaacaatt tgtaaaagca 540 tttgttacaa atatcggtggtcgtacaagt cactcagcta tcatggcacg tacacttgag 600 atcgctgcgg tacttggaacaaatgatatt acaaaacgtg ttaaagatgg tgatgtgatt 660 gccgttaatg gtatcactggtgaagtgatt atcgatccaa gcgaagatca agtacttgct 720 tttaaagaag ctggtgcggcttatgccaaa caaaaagcag agtggtctct ccttaaagat 780 gcgcacactg aaacagctgatggcaaacac tttgaattgg ctgctaatat cggtacgcct 840 aaagacgttg aaggtgttaatgacacagct gatggcaaac actttgaatt ggctgctaat 900 atcggtacgc ctaaagacgttgaaggtgtt aatgacaatg gtgctgaagc tgttggcctt 960 taccgtactg agttcttgtacatggattct caagacttcc caactgaaga cgaacaatac 1020 gaagcttaca aggcagtgcttgaaggcatg aatggcaaac ctgttgtggt tcgtacgatg 1080 gatattggtg gcgacaaggaacttccttac tttgaccttc caaaagaaat gaatccattc 1140 cttggtttcc gtgctcttcgtatttccatc tctgaaactg gggatgccat gttccgcaca 1200 caaatgcgtg cgcttcttcgtgcctctgtt cacggacaac ttcgtattat gttcccaatg 1260 gttgcgcttc ttaaagaattccgtgctgca aaagcaatct ttgacgaaga aaaagcaaac 1320 ttgcttgcag aaggcgttgcggttgctgat gacatccaag ttggtatcat gattgagatt 1380 cctgcagctg ctatgcttgcagaccaattt gctaaggaag ttgatttctt ctcaattgga 1440 acaaacgacc ttatccaatacactatggca gcagaccgta tgaacgaaca agtatcatac 1500 ctttaccaac catacaacccatcaatatta cgtttgatca acaatgtgat caaagcagcg 1560 cacgctgaag gtaaatgggcaggtatgtgt ggtgagatgg caggtgacca acaagctgtt 1620 ccacttcttg tcggaatgggcttggatgag ttttctatgt cagcaacttc agtacttcgt 1680 acgcgtagtt taatgaagaaacttgactct gctaagatgg aagaatatgc aaatcgtgcg 1740 cttacagaat gttcaacagcagaagaagtt cttgaacttt ctaaagaata cgtttctgaa 1800 gat 1803 6 629 PRTGroup A Streptococcus ptsI sequence from Oklahoma University M1 strain 6Met Glu Thr Thr Glu Met Glu Thr Leu Lys Gly Ile Ala Ala Ser Asp 1 5 1015 Gly Val Ala Val Ala Lys Ala Tyr Leu Leu Val Gln Pro Asp Leu Ser 20 2530 Phe Glu Thr Val Thr Val Ala Asp Thr Asn Ala Glu Glu Ala Arg Leu 35 4045 Asp Val Ala Leu Gln Ala Ala Gln Asp Glu Leu Ser Val Ile Arg Glu 50 5560 Asn Ala Val Glu Ser Leu Gly Glu Glu Ala Ala Ala Val Phe Asp Ala 65 7075 80 His Leu Met Glu Thr Val Leu Ala Asp Pro Glu Met Glu Thr Ile Ser 8590 95 Gln Val Lys Glu Thr Ile Arg Ala Lys Gln Thr Asn Ala Glu Thr Gly100 105 110 Leu Lys Glu Val Thr Asp Met Glu Thr Phe Ile Thr Ile Phe GluGly 115 120 125 Met Glu Thr Glu Asp Asn Pro Tyr Met Glu Thr Gln Glu ArgAla Ala 130 135 140 Asp Ile Arg Asp Val Ala Lys Arg Val Leu Ala His LeuLeu Gly Val 145 150 155 160 Lys Leu Pro Asn Pro Ala Thr Ile Asn Glu GluSer Ile Val Ile Ala 165 170 175 His Asp Leu Thr Pro Ser Asp Thr Ala GlnLeu Asn Lys Gln Phe Val 180 185 190 Lys Ala Phe Val Thr Asn Ile Gly GlyArg Thr Ser His Ser Ala Ile 195 200 205 Met Glu Thr Ala Arg Thr Leu GluIle Ala Ala Val Leu Gly Thr Asn 210 215 220 Asp Ile Thr Lys Arg Val LysAsp Gly Asp Val Ile Ala Val Asn Gly 225 230 235 240 Ile Thr Gly Glu ValIle Ile Asp Pro Ser Glu Asp Gln Val Leu Ala 245 250 255 Phe Lys Glu AlaGly Ala Ala Tyr Ala Lys Gln Lys Ala Glu Trp Ser 260 265 270 Leu Leu LysAsp Ala His Thr Glu Thr Ala Asp Gly Lys His Phe Glu 275 280 285 Leu AlaAla Asn Ile Gly Thr Pro Lys Asp Val Glu Gly Val Asn Asp 290 295 300 AsnGly Ala Glu Ala Val Gly Leu Tyr Arg Thr Glu Phe Leu Tyr Met 305 310 315320 Glu Thr Asp Ser Gln Asp Phe Pro Thr Glu Asp Glu Gln Tyr Glu Ala 325330 335 Tyr Lys Ala Val Leu Glu Gly Met Glu Thr Asn Gly Lys Pro Val Val340 345 350 Val Arg Thr Met Glu Thr Asp Ile Gly Gly Asp Lys Glu Leu ProTyr 355 360 365 Phe Asp Leu Pro Lys Glu Met Glu Thr Asn Pro Phe Leu GlyPhe Arg 370 375 380 Ala Leu Arg Ile Ser Ile Ser Glu Thr Gly Asp Ala MetGlu Thr Phe 385 390 395 400 Arg Thr Gln Met Glu Thr Arg Ala Leu Leu ArgAla Ser Val His Gly 405 410 415 Gln Leu Arg Ile Met Glu Thr Phe Pro MetGlu Thr Val Ala Leu Leu 420 425 430 Lys Glu Phe Arg Ala Ala Lys Ala ValPhe Asp Glu Glu Lys Ala Asn 435 440 445 Leu Leu Ala Glu Gly Val Ala ValAla Asp Asp Ile Gln Val Gly Ile 450 455 460 Met Glu Thr Ile Glu Ile ProAla Ala Ala Met Glu Thr Leu Ala Asp 465 470 475 480 Gln Phe Ala Lys GluVal Asp Phe Phe Ser Ile Gly Thr Asn Asp Leu 485 490 495 Ile Gln Tyr ThrMet Glu Thr Ala Ala Asp Arg Met Glu Thr Asn Glu 500 505 510 Gln Val SerTyr Leu Tyr Gln Pro Tyr Asn Pro Ser Ile Leu Arg Leu 515 520 525 Ile AsnAsn Val Ile Lys Ala Ala His Ala Glu Gly Lys Trp Ala Gly 530 535 540 MetGlu Thr Cys Gly Glu Met Glu Thr Ala Gly Asp Gln Gln Ala Val 545 550 555560 Pro Leu Leu Val Gly Met Glu Thr Gly Leu Asp Glu Phe Ser Met Glu 565570 575 Thr Ser Ala Thr Ser Val Leu Arg Thr Arg Ser Leu Met Glu Thr Lys580 585 590 Lys Leu Asp Ser Ala Lys Met Glu Thr Glu Glu Tyr Ala Asn ArgAla 595 600 605 Leu Thr Glu Cys Ser Thr Ala Glu Glu Val Leu Glu Leu SerLys Glu 610 615 620 Tyr Val Ser Glu Asp 625 7 1379 DNA Group AStreptcoccus ptsI sequence from isolate no. 6 7 gttatccgtg aaaatgcagtagaaagctta ggtgaagaag cagcagccgt ttttgatgcc 60 catttgatgg ttcttgctgatccagaaatg atcagccagg ttaaagaaac gattcgcgca 120 aaacaaacga atgcagaaacaggtcttaaa gaagtgactg acatgttcat caccatcttt 180 gaaggcatgg aagataacccatacatgcaa gaacgtgcag cggacatccg cgacgttgca 240 aaacgtgtgt tggctcaccttttaggtgta aaacttccaa atccagctac aatcaatgaa 300 gaatcaatcg ttatcgcacacgatttgaca ccttcagata ctgctcaact taacaaacaa 360 tttgtaaaag catttgttacaaatatcggt ggtcgtacaa gtcactcagc tatcatggca 420 cgtacacttg agatcgctgcggtacttgga acaaatgata ttacaaaacg tgttaaagat 480 ggtgatgtga ttgccgttaatggtatcact ggtgaagtga ttatcgatcc aagcgaagat 540 caagtacttg ctgcagagtggtctctcctt aaagatgcgc acactgaatt taaagaagct 600 ggtgcggctt atgccaaacaaaaaacagct gatggcaaac actttgaatt ggctgctaat 660 atcggtacgc ctaaagacgttgaaggtgtt aatgacaatg gtgctgaagc tgttggcctt 720 taccgtactg agttcttgtacatggattct caagacttcc caactgaaga cgaacaatac 780 gaagcttaca aggcagtgcttgaaggcatg aatggcaaac ctgttgtggt tcgtacgatg 840 gatattggtg gcgacaaggaacttccttac tttgaccttc caaaagaaat gaatccattc 900 cttggtttcc gtgctcttcgtatttccatc tctgaaactg gggatgccat gttccgcaca 960 caaatgcgtg cgcttcttcgtgcctctgtt cacggacaac ttcgtattat gttcccaatg 1020 gttgcgcttc ttaaagaattccgtgctgca aaagcaatct ttgacgaaga aaaagcaaac 1080 ttgcttgcag aaggcgttgcggttgctgat gacatccaag ttggtatcat gattgagatt 1140 cctgcagctg ctatgcttgcagaccaattt gctaaggaag ttgatttctt ctcaattgga 1200 acaaacgacc ttatccaatacactatggca gcagaccgta tgaacgaaca agtatcatac 1260 ctttaccaac catacaacccatcaatatta cgtttgatca acaatgtgat caaagcagcg 1320 cacgctgaag gtaaatgggcaggtatgtgt ggtgagatgg caggtgacca acaagctgt 1379 8 1384 DNA Group AStreptococcus ptsI sequence from isolate no. 5 8 tgttatccgt gaaaatgcagtagaaagctt aggtgaagaa gcagcagccg tttttgatgc 60 ccatttgatg gttcttgctgatccagaaat gatcagccag gttaaagaaa cgattcgcgc 120 aaaacaaacg aatgcagaaacaggtcttaa agaagtgact gacatgttca tcaccatctt 180 tgaaggcatg gaagataacccatacatgca agaacgtgca gcggacatcc gcgacgttgc 240 aaaacgtgtg ttggctcaccttttaggtgt aaaacttcca aatccagcta caatcaatga 300 agaatcaatc gttatcgcacacgatttgac accttcagat actgctcaac ttaacaaaca 360 atttgtaaaa gcatttgttacaaatatcgg tggtcgtaca agtcactcag ctatcatggc 420 acgtacactt gagatcgctgcggtacttgg aacaaatgat attacaaaac gtgttaaaga 480 tggtgatgtg attgccgttaatggtatcac tggtgaagtg attatcgatc caagcgaaga 540 tcaagtactt gcttttaaagaagctggtgc ggcttatgcc aaacaaaaag cagagtggtc 600 tctccttaaa gatgcgcacactgaaacagc tgatggcaaa cactttgaat tggctgctaa 660 tatcggtacg cctaaagacgttgaaggtgt taatgacaat ggcgctgaag ctgttggcct 720 ttaccgtact gagttcttgtacatggattc tcaagacttc ccaactgaag acgaacaata 780 cgaagcttac aaagcagtgcttgaaggcat gaatggcaaa cctgttgtgg ttcgtacaat 840 ggatattggt ggagataaggaacttcctta ctttgacctt ccaaaagaaa tgaatccatt 900 ccttggtttc cgtgctcttcgtatttccat ctctgaaact ggggatgcca tgttccgcac 960 acaaatgcgt gcgcttcttcgcgcctctgt tcacggacaa cttcgtatca tgttcccaat 1020 ggtagcactt cttaaagaattccgtgctgc aaaagcaatc tttgatgaag aaaaagcaaa 1080 cttgcttgca gaaggcgttgcggttgctga tgacatccaa gttggtatca tgattgagat 1140 tcctgcagct gctatgcttgcagaccaatt tgctaaggaa gttgatttct tctcaattgg 1200 aacaaacgac cttatccaatacactatggc agcagaccgt atgaacgaac aagtatcata 1260 cctttaccaa ccatacaacccatcaatatt acgtttgatc aacaatgtga tcaaagcagc 1320 gcacgctgaa ggtaaatgggcaggtatgtg tggtgagatg gcaggtgacc aacaagctgt 1380 tcca 1384 9 1385 DNAGroup A Streptococcus ptsI sequence from isolate no. 7 9 tgttatccgtgaaaatgcag tagaaagctt aggtgaagaa gcagcagccg tttttgatgc 60 ccatttgatggttcttgctg atccagaaat gatcagccag gttaaagaaa cgattcgcgc 120 aaaacaaacgaatgcagaaa caggtcttaa agaagtgact gacatgttca tcaccatctt 180 tgaaggcatggaagataacc catacatgca agaacgtgca gcggacatcc gcgacgttgc 240 aaaacgtgtgttggctcacc ttttaggtgt aaaacttcca aatccagcta caatcaatga 300 agaatcaatcgttatcgcac acgatttgac accttcagat actgctcaac ttaacaaaca 360 atttgtaaaagcatttgtta caaatatcgg tggtcgtaca agtcactcag ctatcatggc 420 acgtacacttgagatcgctg cggtacttgg aacaaatgat attacaaaac gtgttaaaga 480 tggtgatgtgattgccgtta atggtatcac tggtgaagtg attatcgatc caagcgaaga 540 tcaagtacttgcttttaaag aagctggtgc ggcttatgcc aaacaaaaag cagagtggtc 600 tctccttaaagatgcgcaca ctgaaacagc tgatggcaaa cactttgaat tggctgctaa 660 tatcggtacgcctaaagacg ttgaaggtgt taatgacaat ggtgctgaag ctgttggcct 720 ttaccgtactgagttcttgt acatggattc tcaagacttc ccaactgaag acgaacaata 780 cgaagcttacaaggcagtgc ttgaaggcat gaatggcaaa cctgttgtgg ttcgtacgat 840 ggatattggtggcgacaagg aacttcctta ctttgacctt ccaaaagaaa tgaatccatt 900 ccttggtttccgtgctcttc gtatttccat ctctgaaact ggggacgcca tgttccgcac 960 acaaatacgtgcgcttcttc gcgcctctgt tcacggacaa cttcgtatta tgttcccaat 1020 ggttgcgcttcttaaagaat tccgtgctgc aaaagcagtc tttgatgaag aaaaagcaaa 1080 cttgcttgcagaaggcgttg cggttgctga tgacatccaa gttggtatca tgattgagat 1140 tcctgcagctgctatgcttg cagaccaatt tgctaaggaa gttgatttct tctcaattgg 1200 aacaaacgaccttatccaat acactatggc agcagaccgt atgaacgaac aagtatcata 1260 cctttaccaaccatacaacc catcaatatt acgtttgatc aacaatgtga tcaaagcagc 1320 gcacgctgaaggtaaatggg caggtatgtg tggtgagatg gcaggtgacc aacaagctgt 1380 tccac 138510 1384 DNA Group A Streptococcus ptsI sequence from isolate no. 8 10gttatccgtg aaaatgcagt agaaagctta ggtgaagaag cagcagccgt ttttgatgcc 60catttgatgg ttcttgctga tccagaaatg atcagccagg ttaaagaaac gattcgcgca 120aaacaaacga atgcagaaac aggtcttaaa gaagtgactg acatgttcat caccatcttt 180gaaggcatgg aagataaccc atacatgcaa gaacgtgcag cggacatccg cgacgttgca 240aaacgtgtgt tggctcacct tttaggtgta aaacttccaa atccagctac aatcaatgaa 300gaatcaatcg ttatcgcaca cgatttgaca ccttcagata ctgctcaact taacaaacaa 360tttgtaaaag catttgttac aaatatcggt ggtcgtacaa gtcactcagc tatcatggca 420cgtacacttg agatcgctgc ggtacttgga acaaatgata ttacaaaacg tgttaaagat 480ggtgatgtga ttgccgttaa tggtatcact ggtgaagtga ttatcgatcc aagcgaagat 540caagtacttg cttttaaaga agctggtgcg gcttatgcca aacaaaaagc agagtggtct 600ctccttaaag atgcgcatac tgaaacagct gatggcaaac actttgaatt ggctgctaat 660atcggtacac ctaaagacgt tgaaggtgtt aatgacaatg gcgctgaagc tgttggcctt 720taccgtactg agttcttgta catggattct caagacttcc caactgaaga cgaacaatac 780gaagcttaca aggcagtgct tgaaggcatg aatggcaaac ctgttgtggt tcgtacgatg 840gatattggtg gcgacaagga acttccttac tttgaccttc caaaagaaat gaatccattc 900cttggtttcc gtgctcttcg tatttccatc tctgaaactg gggatgccat gttccgcaca 960caaatgcgtg cgcttcttcg tgcctctgtt cacggacaac ttcgtattat gttcccaatg 1020gttgcccttc ttaaagaatt ccgtgctgca aaagcagtct ttgatgaaga aaaagcaaac 1080ttgcttgcag aaggcgttgc ggttgctgat gacatccaag ttggtatcat gattgagatt 1140cctgcagctg ctatgcttgc agaccaattt gctaaggaag ttgatttctt ctcaattgga 1200acaaacgacc ttatccaata cactatggca gcagaccgta tgaacgaaca agtatcatac 1260ctttaccaac catacaaccc atcaatatta cgtttgatca acaatgtgat caaagcagcg 1320cacgctgaag gtaaatgggc aggtatgtgt ggtgagatgg caggtgacca acaagctgtt 1380ccac 1384 11 1390 DNA Group A Streptococcus ptsI sequence from isolateno. 9 11 caagacgagc tttctgttat ccgtgaaaat gcagtagaaa gcttaggtgaagaagcagca 60 gccgtttttg atgcccattt gatggttctt gctgatccag aaatgatcagccaggttaaa 120 gaaacgattc gcgcaaaaca aacgaatgca gaaacaggtc ttaaagaagtgactgacatg 180 ttcatcacca tctttgaagg catggaagat aacccataca tgcaagaacgcgcagcggac 240 atccgcgacg ttgcaaaacg tgtgttggct caccttttag gtgtaaaacttccaaatcca 300 gctacaatca atgaagaatc aatcgttatc gcacacgatt tgacaccttcagatactgct 360 caacttaaca aacaatttgt aaaagcattt gttacaaata tcggtggtcgtacaagtcac 420 tcagctatca tggcacgtac acttgagatc gctgcggtac ttggaacaaatgatattaca 480 aaacgtgtta aagatggtga tgtgattgcc gttaatggta tcactggtgaagtgattatc 540 gatccaagcg aagatcaagt acttgctttt aaagaagctg gtgcggcttatgccaaacaa 600 aaagcagagt ggtctctcct taaagatgcg catactgaaa cagctgatggcaaacacttt 660 gaattggctg ctaatatcgg tacacctaaa gacgttgaag gtgttaatgacaatggcgct 720 gaagctgttg gcctttaccg tactgagttc ttgtacatgg attctcaagacttcccaact 780 gaagacgaac aatacgaagc ttacaaggca gtgcttgaag gcatgaatggcaaacctgtt 840 gtggttcgta cgatggatat tggtggcgac aaggaacttc cttactttgaccttccaaaa 900 gaaatgaatc cattccttgg tttccgtgct cttcgtattt ccatctctgaaactggggat 960 gccatgttcc gcacacaaat gcgtgcgctt cttcgtgcct ctgttcacggacaacttcgt 1020 attatgttcc caatggttgc ccttcttaaa gaattccgtg ctgcaaaagcagtctttgat 1080 gaagaaaaag caaacttgct tgcagaaggc gttgcggttg ctgatgacatccaagttggt 1140 atcatgattg agattcctgc agctgctatg cttgcagacc aatttgctaaggaagttgat 1200 ttcttctcaa ttggaacaaa cgaccttatc caatacacta tggcagcagaccgtatgaac 1260 gaacaagtat cataccttta ccaaccatac aacccatcaa tattacgtttgatcaacaat 1320 gtgatcaaag cagcgcacgc tgaaggtaaa tgggcaggta tgtgtggtgagatggcaggt 1380 gaccaacaag 1390 12 1323 DNA Group A Streptococcus ptsIsequence from isolate no. 2 12 cagtagaaag cttaggtgaa gaagcagcagccgtttttga tgcccatttg atggttcttg 60 ctgatccaga aatgattagc caggttaaagaaacgattcg cgcaaaacaa acgaatgcag 120 aaacaggtct taaagaagtg actgacatgttcatcaccat ctttgaaggc atggaagata 180 acccatacat gcaagaacgt gcagcggacattcgcgacgt tgcaaaacgt gtgttggctc 240 accttttagg tgtaaaactt ccaaatccagctacaatcaa tgaagaatca atcgttatcg 300 cacacgattt gacaccttca gatactgctcaacttaacaa acaatttgta aaagcatttg 360 ttacaaatat cggtggtcgt acaagtcactcagctatcat ggcacgtaca cttgagatcg 420 ctgcggtact tggaacaaat gatattacaaaacgtgttaa agatggtgat gtgattgccg 480 ttaatggtat cactggtgaa gtgattatcgatccaagcga agatcaagta cttgctttta 540 aagaagctgg tgcggcttat gccaaacaaaaagcagagtg gtctctcctt aaagatgcgc 600 atactgaaac agctgatggc aaacactttgaattggctgc taatatcggt acgcctaaag 660 acgttgaagg tgttaatgac aatggcgctgaagctgttgg cctttaccgt actgagttct 720 tgtacatgga ttctcaagac ttcccaactgaagacgaaca atacgaagct tacaaggcag 780 tgcttgaagg catgaatggc aaacctgtcgtggttcgtac gatggatatt ggtggcgaca 840 aggaacttcc ttactttgac cttccaaaagaaatgaatcc attccttggt ttccgtgctc 900 ttcgtatttc catctctgaa actggggatgccatgttccg cacacaaatg cgtgcgcttc 960 ttcgtgcctc tgttcacgga caacttcgtattatgttccc aatggttgcg cttcttaaag 1020 aattccgtgc tgcaaaagca ggcgttgcggttgctgatga cattcaagtt ggtatcatga 1080 ttgagattcc tgcagctgct atgcttgcagaccaatttgc taaggaagtt gatttcttct 1140 caattggaac aaacgacctt atccaatacactatggcagc agaccgtatg aacgaacaag 1200 tatcatacct ttaccaacca tacaacccatcaatattacg tttgatcaac aatgtgatca 1260 aagcagcgca cgctgaaggt aaatgggcaggtatgtgtgg tgagatggca ggtgaccaac 1320 aag 1323 13 1379 DNA Group AStreptococcus ptsI sequence from isolate no. 4 13 gttatccgtg aaaatgcagtagaaagctta ggtgaagaag cagcagccgt ttttgatgcc 60 catttgatgg ttcttgctgatccagaaatg attagccagg ttaaagaaac gattcgcgca 120 aaacaaacga atgcagaaacaggtcttaaa gaagtgactg acatgttcat caccatcttt 180 gaaggcatgg aagataacccatacatgcaa gaacgtgcag cggacattcg cgacgttgca 240 aaacgtgtgt tggctcaccttttaggtgta aaacttccaa atccagctac aatcaatgaa 300 gaatccatcg ttatcgcacacgatttgaca ccttcagata ctgctcaact taacaaacaa 360 tttgtaaaag catttgttacaaatatcggt ggtcgtacaa gtcactcagc tatcatggca 420 cgtacacttg agatcgctgcggtacttgga acaaatgata ttacaaaacg tgttaaagat 480 ggtgatgtga ttgccgttaatggtatcact ggtgaagtga ttatcgatcc aagcgaagat 540 caagtacttg cttttaaagaagctggtgcg gcttatgcca aacaaaaagc agagtggtct 600 ctccttaaag atgcgcatactgaaacagct gatggcaaac actttgaatt ggctgctaat 660 atcggtacac ctaaagacgttgaaggtgtt aatggcaatg gcgctgaagc tgttggcctt 720 taccgtactg agttcttgtacatggattct caagacttcc caactgaaga cgaacaatac 780 gaagcttaca aggcagtgcttgaaggcatg aatggcaaac ctgttgtggt tcgtacgatg 840 gatattggtg gcgacaaggaacttccttac tttgaccttc caaaagaaat gaatccattc 900 cttggtttcc gtgctcttcgtatttccatc tctgaaactg gggatgccat gttccgcaca 960 caaatgcgtg cgcttcttcgtgcctctgtt cacggacaac ttcgtattat gttcccaatg 1020 gttgcccttc ttaaagaattccgtgctgca aaagcagtct ttgatgaaga aaaagcaaac 1080 ttgcttgcag aaggcgttgcggttgctgat gacatccaag ttggtatcat gattgagatt 1140 cctgcagctg ctatgcttgcagaccaattt gctaaggaag ttgatttctt ctcaattgga 1200 acaaacgacc ttatccaatacactatggca gcagaccgta tgaacgaaca agtatcatac 1260 ctttaccaac catacaacccatcaatatta cgtttgatca acaatgtgat caaagcagcg 1320 cacgctgaag gtaaatgggcaggtatgtgt ggtgagatgg caggtgacca acaagctgt 1379 14 1378 DNA Group AStreptococcus ptsI sequence from isolate no. 10 14 gttatccgtg aaaatgcagtagaaagctta ggtgaagaag cagcagccgt ttttgatgcc 60 catttaatgg ttcttgctgatccagaaatg atcagccagg ttaaagaaac gattcgcgca 120 aaacaaacga atgcagaaacaggtcttaaa gaagtgactg acatgttcat caccatcttt 180 gaaggcatgg aagataacccatacatgcaa gaacgtgcag cggacatccg cgacgttgca 240 aagcgtgtgt tggctcaccttttaggtgta aaacttccaa atccagctac aatcaatgaa 300 gaatcaatcg ttatcgcacacgatttgaca ccttcagata ctgctcaact taacaaacaa 360 tttgtaaaag catttgttacaaatatcggt ggtcgtacaa gtcactcagc tatcatggca 420 cgtacacttg agatcgctgcggtacttgga acaaatgata ttacaaaacg tgttaaagat 480 ggtgatgtga ttgccgttaatggtatcact ggtgaagtga ttatcgatcc aagcgaagat 540 caagtacttg cttttaaagaagctggtgcg gcttatgcca aacaaaaagc agagtggtct 600 ctccttaaag atgcgcatactgaaacagct gatggcaaac actttgaatt ggctgctaat 660 atcggtacgc ctaaagacgttgaaggtgtt aatgacaatg gcgctgaagc tgttggcctt 720 taccgtactg agttcttgtacatggattct caagacttcc caactgaaga cgaacaatac 780 gaagcttaca aggcagtgcttgaaggcatg aatggcaaac ctgttgtggt tcgtacgatg 840 gatattggtg gcgacaaggaacttccttac tttgaccttc caaaagaaat gaatccattc 900 cttggtttcc gtgctcttcgtatttccatc tctgaaactg gggatgccat gttccgcaca 960 caaatgcgtg cgcttcttcgtgcctctgtt cacggacaac ttcgtattat gttcccaatg 1020 gttgcccttc ttaaagaattccgtgctgca aaagcaatct ttgatgaaga aaaagcaaac 1080 ttgcttgcag aaggcgttgcggttgctgat gacatccaag ttggtatcat gattgagatt 1140 cctgcagctg ctatgcttgcagaccaattt gctaaggaag ttgatttctt ctcaattgga 1200 acaaacgacc ttatccaatacactatggca gcagatcgta tgaacgaaca agtatcatac 1260 ctttaccaac catacaacccatcaatatta cgtttgatca acaatgtgat caaagcagcg 1320 cccgctgaag gtaaatgggcaggtatgtgt ggtgagatgg caggtgacca acaagctg 1378 15 1393 DNA Group AStreptococcus ptsI sequence from isolate no. 3 15 tgttatccgt gaaaatgcagtagaaagctt aggtgaagaa gcagcagccg tttttgatgc 60 ccatttaatg gttcttgctgatccagaaat gatcagccag gttaaagaaa cgattcgcgc 120 aaaacaaacg aatgcagaaacaggtcttaa agaagtgact gacatgttca tcaccatctt 180 tgaaggcatg gaagataacccatacatgca agaacgtgca gcggacatcc gcgacgttgc 240 aaagcgtgtg ttggctcaccttttaggtgt aaaacttcca aatccagcta caatcaatga 300 agaatcaatc gttatcgcacacgatttgac accttcagat actgctcaac ttaacaaaca 360 atttgtaaaa gcatttgttacaaatatcgg tggtcgtaca agtcactcag ctatcatggc 420 acgtacactt gagatcgctgcggtacttgg aacaaatgat attacaaaac gtgttaaaga 480 tggtgatgtg attgccgttaatggtatcac tggtgaagtg attatcgatc caagcgaaga 540 tcaagtactt gcttttaaagaagctggtgc ggcttatgcc aaacaaaaag cagagtggtc 600 tctccttaaa gatgcgcatactgaaacagc tgatggcaaa cactttgaat tggctgctaa 660 tatcggtacg cctaaagacgttgaaggtgt taatgacaat ggcgctgaag ctgttggcct 720 ttaccgtact gagttcttgtacatggattc tcaagacttc ccaactgaag acgaacaata 780 cgaagcttac aaggcagtgcttgaaggcat gaatggcaaa cctgttgtgg ttcgtacgat 840 ggatattggt ggcgacaaggaacttcctta ctttgacctt ccaaaagaaa tgaatccatt 900 ccttggtttc cgtgctcttcgtatttccat ctctgaaact ggggatgcca tgttccgcac 960 acaaatgcgt gcgcttcttcgtgcctctgt tcacggacaa cttcgtatta tgttcccaat 1020 ggttgccctt cttaaagaattccgtgctgc aaaagcaatc tttgatgaag aaaaagcaaa 1080 cttgcttgca gaaggcgttgcggttgctga tgacatccaa gttggtatca tgattgagat 1140 tcctgcagct gctatgcttgcagaccaatt tgctaaggaa gttgatttct tctcaattgg 1200 aacaaacgac cttatccaatacactatggc agcagatcgt atgaacgaac aagtatcata 1260 cctttaccaa ccatacaacccatcaatatt acgtttgatc aacaatgtga tcaaagcagc 1320 gcacgctgaa ggtaaatgggcaggtatgtg tggtgagatg gcaggtgacc aacaagctgt 1380 tccacttctt gtc 1393 161379 DNA Group A Streptococcus ptsI sequence from isolate no. 1 16tgttatccgt gaaaatgcag tagaaagctt aggtgaagaa gcagcagccg tttttgatgc 60ccatttgatg gttcttgctg atccagaaat gatcagccag gttaaagaaa cgattcgcgc 120aaaacaaacg aatgcagaaa caggtcttaa agaagtgact gacatgttca tcaccatctt 180tgaaggcatg gaagataacc catacatgca agaacgtgca gcggacatcc gcgacgttgc 240aaaacgtgtg ttggctcacc ttttaggtgt aaaacttcca aatccagcta caatcaatga 300agaatcaatc gttatcgcac acgatttgac accttcagat actgctcaac ttaacaaaca 360atttgtaaaa gcatttgtta caaatatcgg tggtcgtaca agtcactcag ctatcatggc 420acgtacactt gagatcgctg cggtacttgg aacaaatgat attacaaaac gtgttaaaga 480tggtgatgtg attgccgtta atggtatcac tggtgaagtg attatcgatc caagcgaaga 540tcaagtactt gcttttaaag aagctggtgc ggcttatgcc aaacaaaaag cagagtggtc 600tctccttaaa gatgcgcata ctgaaacagc tgatggcaaa cactttgaat tggctgctaa 660tatcggtacg cctaaagacg ttgaaggtgt taatgacaat ggcgctgaag ctgttggcct 720ttaccgtact gagttcttgt acatggattc tcaagacttc ccaactgaag acgaacaata 780cgaagcttac aaggcagtgc ttgaaggcat gaatggcaaa cctgttgtgg ttcgtacaat 840ggatattggt ggagataagg aacttcctta ctttgacctt ccaaaagaaa tgaacccatt 900cctcggtttc cgtgctcttc gtatctcaat ctctgaaact ggggatgcca tgttccgcac 960acaaatgcgt gcgcttcttc gcgcctctgt tcacggacaa cttcgtatca tgttcccaat 1020ggtagcactt cttaaagaat tccgtgctgc aaaagcaatc tttgacgaag aaaaagcaaa 1080cttgcttgca gaaggcgttg cggttgctga tgacatccaa gttggtatca tgattgagat 1140tcctgcagct gctatgcttg cagaccaatt tgctaaggaa gttgatttct tctcaattgg 1200aacaaacgac cttatccaat acactatggc agcagaccgt atgaacgaac aagtatcata 1260cctttaccaa ccatacaacc catcaatatt acgtttgatc aacaatgtga tcaaagcagc 1320gcacgctgaa ggtaaatggg caggtatgtg tggtgagatg gcaggtgacc aacaagctg 1379 171447 DNA Group A Streptococcus ptsI sequence from isolate no. 11 17tgttatccgt gaaaatgcag tagaaagctt aggtgaagaa gcagcagccg tttttgatgc 60ccatttgatg gttcttgctg atccagaaat gatcagccag gttaaagaaa cgattcgcgc 120aaaacaaacg aatgcagaaa caggtcttaa agaagtgact gacatgttca tcaccatctt 180tgaaggcatg gaagataacc catacatgca agaacgcgca gcggacatcc gcgacgttgc 240aaaacgtgtg ttggctcacc ttttaggtgt aaaacttcca aatccagcta caatcaatga 300agaatcaatc gttatcgcac acgatttgac accttcagat actgctcaac ttaacaaaca 360atttgtaaaa gcatttgtta caaatatcgg tggtcgtaca agtcactcag ctatcatggc 420acgtacactt gagatcgctg cggtacttgg aacaaatgat attacaaaac gtgttaaaga 480tggtgatgtg attgccgtta atggtatcac tggtgaagtg attatcgatc caagcgagga 540tcaagtactt gcttttaaag aagctggtgc ggcttatgcc aaacaaaaag cagagtggtc 600tctccttaaa gatgcgcata ctgaaacagc tgatggcaaa cactttgaat tggctgctaa 660tatcggtacg cctaaagatg ttgaaggtgt taatgacaat ggtgctgaag ctgttggcct 720ttaccgtact gagttcttgt acatggattc tcaagacttc ccaactgaag acgaacaata 780cgaagcttac aaggcagtgc ttgaaggcat gaatggcaaa cctgttgtgg ttcgtacaat 840ggatattggt ggagataagg aacttcctta ctttgacctt ccaaaagaaa tgaacccatt 900cctcggtttc cgtgctcttc gtatctcaat ctctgaaact ggggatgcca tgttccgcac 960acaaatgcgt gcgcttcttc gtgcctctgt tcacggacaa cttcgtatca tgttcccaat 1020ggtagcactt cttaaagaat tccgtgctgc aaaagcaatc tttgacgaag aaaaagcaaa 1080cttgcttgca gaaggcgttg cggttgctga tgacatccaa gttggtatca tgattgagat 1140tcctgcagct gctatgcttg cagaccaatt tgctaaggaa gttgatttct tctcaattgg 1200aacaaacgac cttatccaat acactatggc agcagaccaa tttgctaagg aagttgattt 1260cttctcaatt ggaacaaacg accttatcca atacactatg gcagcagacc gtatgaacga 1320acaagtatca tacctttacc aaccatacaa cccatcaata ttacgtttga tcaacaatgt 1380gatcaaagca gcgcacgctg aaggtaaatg ggcaggtatg tgtggtgaga tggcaggtga 1440ccaacaa 1447

What is claimed is:
 1. A method for detecting the presence or absence ofGroup A Streptococcus (GAS) in a biological sample from an individual,said method comprising: performing at least one cycling step, wherein acycling step comprises an amplifying step and a hybridizing step,wherein said amplifying step comprises contacting said sample with apair of ptsI primers to produce a ptsI amplification product if a GASptsI nucleic acid molecule is present in said sample, wherein saidhybridizing step comprises contacting said sample with a pair of ptsIprobes, wherein the members of said pair of ptsI probes hybridize tosaid amplification product within no more than five nucleotides of eachother, wherein a first ptsI probe of said pair of ptsI probes is labeledwith a donor fluorescent moiety and wherein a second ptsI probe of saidpair of ptsI probes is labeled with a corresponding acceptor fluorescentmoiety; and detecting the presence or absence of fluorescence resonanceenergy transfer (FRET) between said donor fluorescent moiety of saidfirst ptsI probe and said acceptor fluorescent moiety of said secondptsI probe, wherein the presence of FRET is indicative of the presenceof GAS in said biological sample, and wherein the absence of FRET isindicative of the absence of GAS in said biological sample.
 2. Themethod of claim 1, wherein said pair of ptsI primers comprises a firstptsI primer and a second ptsI primer, wherein said first ptsI primercomprises the sequence 5′-AAA TGC AGT AGA AAG CTT AGG-3′ (SEQ ID NO: 1),and wherein said second ptsI primer comprises the sequence 5′-TGC ATGTAT GGG TTA TCT TCC-3′ (SEQ ID NO: 2).
 3. The method of claim 1, whereinsaid first ptsI probe comprises the sequence 5′-TTG CTG ATC CAG AAA TGAT-3′ (SEQ ID NO: 3), and wherein said second ptsI probe comprises thesequence 5′-AGC CAG GTT AAA GAA ACG ATT CGC-3′ (SEQ ID NO: 4).
 4. Themethod of claim 1, wherein the members of said pair of ptsI probeshybridize within no more than two nucleotides of each other.
 5. Themethod of claim 1, wherein the members of said pair of ptsI probeshybridize within no more than one nucleotide of each other.
 6. Themethod of claim 1, wherein said donor fluorescent moiety is fluorescein.7. The method of claim 1, wherein said acceptor fluorescent moiety isselected from the group consisting of LC-Red 640, LC-Red 705, Cy5, andCy5.5.
 8. The method of claim 1, wherein said detecting step comprisesexciting said biological sample at a wavelength absorbed by said donorfluorescent moiety and visualizing and/or measuring the wavelengthemitted by said acceptor fluorescent moiety.
 9. The method of claim 1,wherein said detecting comprises quantitating said FRET.
 10. The methodof claim 1, wherein said detecting step is performed after each cyclingstep.
 11. The method of claim 1, wherein said detecting step isperformed in real-time.
 12. The method of claim 1, further comprisingdetermining the melting temperature between one or both of said ptsIprobe(s) and said ptsI amplification product, wherein said meltingtemperature confirms said presence or said absence of said GAS.
 13. Themethod of claim 1, wherein the presence of said FRET within 50 cycles isindicative of the presence of a GAS infection in said individual. 14.The method of claim 1, wherein the presence of said FRET within 40cycles is indicative of the presence of a GAS infection in saidindividual.
 15. The method of claim 1, wherein the presence of said FRETwithin 30 cycles is indicative of the presence of a GAS infection insaid individual.
 16. The method of claim 1, further comprising:preventing amplification of a contaminant nucleic acid.
 17. The methodof claim 16, wherein said preventing comprises performing saidamplification step in the presence of uracil.
 18. The method of claim17, wherein said preventing further comprises treating said biologicalsample with uracil-DNA glycosylase prior to a first amplification step.19. The method of claim 1 wherein said biological sample is selectedfrom the group consisting of throat swabs, tissues and bodily fluids.20. The method of claim 1, wherein said cycling step is performed on acontrol sample.
 21. The method of claim 20, wherein said control samplecomprises said GAS ptsI nucleic acid molecule.
 22. The method of claim1, wherein said cycling step uses a pair of control primers and a pairof control probes, wherein said control primers and said control probesare other than said ptsI primers and said ptsI probes, respectively,wherein a control amplification product is produced if control templateis present in said sample, wherein said control probes hybridize to saidcontrol amplification product.
 23. An article of manufacture,comprising: a pair of ptsI primers; a pair of ptsI probes; and a donorfluorescent moiety and a corresponding fluorescent moiety.
 24. Thearticle of manufacture of claim 23, wherein said pair of ptsI primerscomprises a first ptsI primer and a second ptsI primer, wherein saidfirst ptsI primer comprises the sequence 5′-AAA TGC AGT AGA AAG CTTAGG-3′ (SEQ ID NO: 1), and wherein said second ptsI primer comprises thesequence 5′-TGC ATG TAT GGG TTA TCT TCC-3′ (SEQ ID NO: 2).
 25. Thearticle of manufacture of claim 23, wherein said pair of ptsI probescomprises a first ptsI probe and a second ptsI probe, wherein said firstptsI probe comprises the sequence 5′-TTG CTG ATC CAG AAA TGA T-3′ (SEQID NO: 3), and wherein said second ptsI probe comprises the sequence5′-AGC CAG GTT AAA GAA ACG ATT CGC-3′ (SEQ ID NO: 4).
 26. The article ofmanufacture of claim 23, wherein said pair of ptsI probes comprises afirst ptsI probe labeled with said donor fluorescent moiety and a secondptsI probe labeled with said corresponding acceptor fluorescent moiety.27. The article of manufacture of claim 23, further comprising a packagelabel or package insert having instructions thereon for using said pairof ptsI primers and said pair of ptsI probes to detect the presence orabsence of GAS in a biological sample.
 28. A method for detecting thepresence or absence of GAS in a biological sample from an individual,said method comprising: performing at least one cycling step, wherein acycling step comprises an amplifying step and a hybridizing step,wherein said amplifying step comprises contacting said sample with apair of ptsI primers to produce a ptsI amplification product if a GASptsI nucleic acid molecule is present in said sample, wherein saidhybridizing step comprises contacting said sample with a ptsI probe,wherein said ptsI probe is labeled with a donor fluorescent moiety and acorresponding acceptor fluorescent moiety; and detecting the presence orabsence of fluorescence resonance energy transfer (FRET) between, saiddonor fluorescent moiety and said acceptor fluorescent moiety of saidptsI probe, wherein the presence or absence of FRET is indicative of thepresence or absence of GAS in said sample.
 29. The method of claim 28,wherein said amplification employs a polymerase enzyme having 5′ to 3′exonuclease activity.
 30. The method of claim 29, wherein said donor andacceptor fluorescent moieties are within no more than 5 nucleotides ofeach other on said probe.
 31. The method of claim 30, wherein saidacceptor fluorescent moiety is a quencher.
 32. The method of claim 28,wherein said ptsI probe comprises a nucleic acid sequence that permitssecondary structure formation, wherein said secondary structureformation results in spatial proximity between said donor and saidacceptor fluorescent moiety.
 33. The method of claim 32, wherein saidacceptor fluorescent moiety is a quencher.
 34. A method for detectingthe presence or absence of GAS in a biological sample from anindividual, said method comprising: performing at least one cyclingstep, wherein a cycling step comprises an amplifying step and adye-binding step,-wherein said amplifying step comprises contacting saidsample with a pair of ptsI primers to produce a ptsI amplificationproduct if a GAS ptsI nucleic acid molecule is present in said sample,wherein said dye-binding step comprises contacting said ptsIamplification product with a nucleic acid binding dye; and detecting thepresence or absence of binding of said nucleic acid binding dye to saidamplification product, wherein the presence of binding is indicative ofthe presence of GAS in said sample, and wherein the absence of bindingis indicative of the absence of GAS in said sample.
 35. The method ofclaim 34, wherein said nucleic acid binding dye is selected from thegroup consisting of SYBRGreenI®, SYBRGold®, and ethidium bromide. 36.The method of claim 35, further comprising determining the meltingtemperature between said ptsI amplification product and said nucleicacid binding dye, wherein said melting temperature confirms saidpresence or absence of said GAS.